Short chain-length specific trans-2-enoyl-CoA reductase (reductase I), which contributed to mitochondrial fatty acid synthesis, was purified about 200-fold from crude extract of mitochondria in Euglena gracilis. It had a molecular weight of 39,000, and consisted of two dissimilar subunits with molecular weights of 15,000 and 25,000. The enzyme utilized crotonyl-CoA as the most active substrate and showed negative cooperativity in the reaction with the substrate. NADH was the sole electron donor. Some divalent cations were inhibitory to the enzyme when incubated with the enzyme prior to the start of the reaction. The reductase apparently contained loosely bound FAD.